This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are studying a mutant Cx50 associated with hereditary congenital cataracts. This mutant, which has a proline to serine substitution at amino acid 88, results in accumulations in the transfected cells in a compartment different from the ER, Golgi or lysosomes. The mutant does not co-localize with the transferrin receptor (used as a marker of the endocytic pathway). We are studying the trafficking of the mutant protein and the mechanism and time course of formation of the accumulations. We generated a construct of the Cx50 mutant with the tetracysteine motif and transfected it into cells. The incubation of the transfected cells with FLASH or ReAsH would allow following the time course and path of formation of these accumulations. Incubation with FLASH and, subsequently, with ReAsH (or vice versa) provides insights as to which pool of protein (old and/or new) participate in the formation of the accumulations. Moreover, we take advantage of the photooxidative properties of the ReAsH reagent that would allow looking at the compartments in which the mutant protein is located by electron microscopy and to make a direct correlation with the live cell images obtained.